Small molecules play important roles across a diversity of biological processes, yet methods for detecting their abundance and activity in living cells are lacking. Common methods for detecting proteins, such as genetic fusion to FRET reporters, N. Mochizuki et al., Nature 411, 1065 (Jun. 28, 2001), to peptides that ligate to chemical probes, G. Gaietta et al., Science 296, 503 (Apr. 19, 2002), or to protein complementation fragments, C. D. Hu, T. K. Kerppola, Nat Biotechnol 21, 539 (May, 2003), are typically inapplicable to the detection of small molecules.
Some metabolite binding proteins undergo dramatic conformational rearrangements upon complex formation with target molecules, so that genetic fusion to FRET pairs or labeling with environmentally sensitive dyes are natural routes to detection, R. Y. Tsien, S. A. Hires, Y. L. Zhu, Proc Natl Acad Sci USA 105, 4411 (Mar. 18, 2008). However, this approach is limited to the minority of well-characterized cases exhibiting allosteric transduction of small-molecule binding events.
Other techniques such as the SNAP, M. A. Brun, K. T. Tan, E. Nakata, M. J. Hinner, K. Johnsson, J Am Chem Soc 131, 5873 (Apr. 29, 2009), and HaloTag, G. V. Los et al., ACS Chem Biol 3, 373 (Jun. 20, 2008), methods involve covalent fusion of target analogs to environmentally-sensitive fluorescent tags that self-label to reporter protein complexes. High target concentrations then compete away the lower affinity analogs causing a shift in reporter fluorescence. Although, in principle, these strategies offer modular approaches to small molecule detection, they require in vitro synthesis with membrane impermeable compounds, prohibiting their use in vivo, R. H. Newman, M. D. Fosbrink, J. Zhang, Chem Rev 111, 3614 (May 11, 2011).